An IFNγ FluoroSpot assay was additionally used for detection of SARS-CoV-2-specific T cells (Mateus et al., 2020 (link)). Briefly, PVDF membrane FluoroSpot plates (Mabtech, Stockholm, Sweden) were coated with 5 μg/mL coating antibody in PBS (anti-IFNγ [clone: 1-D1K]; Mabtech, Stockholm, Sweden) overnight at 4 °C. PBMCs were thawed and resuspended in HR5 medium supplemented with 20 units/mL benzonase nuclease. Cells were counted and seeded into a microtiter plate at 2.5 × 105 cells per well where cells were stimulated with either 1 μg/mL DMSO, 1 μg/mL Spike MP, or 10 μg/mL PHA in HR5 medium for 23–24 h. All stimulations were performed in triplicate. After stimulation, plates were washed with PBS-0.05% Tween20 (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 2 h at room temperature with detection antibody (anti-IFNγ-BAM [clone: 7-B6–1]; Mabtech, Stockholm, Sweden) in PBS-0.1% bovine serum albumin (Rockland Immunochemicals, Gilbertsville, PA, USA). The plates were washed again and incubated for 1 h in the dark with the fluorophore-conjugated antibody solution (anti-BAM-490; Mabtech, Stockholm, Sweden). Plates were read by a Mabtech IRIS FluoroSpot reader (Mabtech, Stockholm, Sweden). Responses are reported as the average number of spot forming cells (SFC)/106 PBMC from the triplicate.
Iris fluorospot reader
Manufactured by Mabtech
Sourced in Sweden
The IRIS FluoroSpot reader is a specialized piece of laboratory equipment designed for the detection and analysis of fluorescently labeled cells. It is capable of quantifying the number of cells secreting specific cytokines or other analytes using the FluoroSpot assay technique.
Automatically generated - may contain errors
Lab products found in correlation
X vivo 15 medium, by Lonza (3 mentions)
Anti bam 490, by Mabtech (2 mentions)
Bovine serum albumin (bsa), by Rockland Immunochemicals (1 mentions)
Fluorospot plates, by Mabtech (1 mentions)
Anti ifnγ clone 1 d1k, by Mabtech (1 mentions)
Pbs 0.05 tween 20, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Eurobio Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
L glutamine, by Lonza (1 mentions)
Nitrocellulose plates, by Merck Group (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Human serum, by Merck Group (1 mentions)
Fluorospot flex, by Mabtech (1 mentions)
Human ifn γ precoated elispot kit, by Dakewe (1 mentions)
Mouse ifn γ precoated elispot kit, by Dakewe (1 mentions)
9 protocols using iris fluorospot reader
1
SARS-CoV-2 Specific T Cell Detection
2
Microbiota-Driven TILs Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TILs (2 × 104 cells per well) were seeded with 5 × 104 irradiated (300 Gy) autologous EBV-transformed B cell line cells per well as APCs primed with bacteria-derived and microbiota-derived peptides (10 µM final concentration). Cells were seeded in X-VIVO 15 medium (Lonza) in human IFNγ/IL-10/IL-17A pre-coated fluorospot plates (FSP-010703-10, Mabtech) and were incubated for 44 h at 37 °C. Before seeding, fluorospot plates were washed three times using PBS and then blocked with RPMI-1640 medium (Sigma-Aldrich) containing 10% FCS (Eurobio) as instructed by the manufacturer. After 44 h of incubation, plates were washed using PBS and incubated with detection antibody, anti-IFNγ monoclonal antibody 7-B6-1-BAM, for 2 h. Next, plates were washed using PBS and incubated with anti-BAM-490 fluorophore conjugate for 1 h. Both detection antibody and fluorophore conjugate were diluted with PBS containing 0.1% BSA (Sigma-Aldrich) as instructed by the manufacturer. Next, plates were washed using PBS and incubated with fluorescence enhancer (Mabtech) for 15 min. Plates were emptied by flicking and kept in the dark for 24 h to completely dry. Finally, spot analysis was performed using the automated Mabtech IRIS fluorospot reader (Mabtech).
3
SARS-CoV-2 Spike Protein Peptide Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following stimulation with either a commercial peptide pool (Mabtech) containing 100 peptides derived from the human SARS-CoV-2 spike protein or an in-house–developed peptide pool of 8 peptides derived from the spike protein21 (link),22 (link) (eTable 1 and eTable 2 in the Supplement ), the number of SFUs was determined with a Mabtech IRIS FluoroSpot reader, and spots were analyzed using Mabtech Apex software, version 1.1. A detailed description of this assay is available in the eMethods in the Supplement .
4
Cryopreserved PBMC Stimulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryopreserved PBMC (5 × 106/sample) were thawed in prewarmed RPMI-1640 media supplemented with L-glutamine + 10% FCS and 300ug DNAse. Thawed PBMCs were rested overnight at 37 °C in X-VIVO™-15 Medium (Lonza) supplemented with 5% human-AB serum. Cells were stimulated with ~1 nmol of peptide pool corresponding to spike of Omicron (B.1.1.529) variant (16-mer peptide pools, overlapping by 10 amino acids (21st century Biochemicals Inc.) on pre-coated human IFN-γ ELISpot plates (Mabtech, Inc.) and developed after 18 hours according to manufacturer instructions. Spots were imaged and counted using Iris FLUOROspot reader (Mabtech).
5
Profiling SARS-CoV-2 Variant-Specific T-Cell Responses
6
IFN-γ ELISPOT Assay for S. aureus Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nitrocellulose plates (Millipore) were coated with anti-human IFN-γ monoclonal antibody (mAb) (Mabtech), at a concentration of 15 µg/mL and incubated overnight at +4°C. Plates were then washed twice and blocked with AIMV medium (Invitrogen). PBMCs were thawed in FBS and washed twice in RPMI 10% FBS. The concentration of viable cells was adjusted to 2 x 106 cells/mL in AIMV. One hundred µL of cell suspension was added per well followed by 100 µL per well of Hla or IsdB S. aureus antigen solution in duplicates, at a final concentration of 10 µg/mL for cell-specific activation. CPI (Immunospot) was used at 5 µg/mL as a positive control. Plates were incubated for 24 hours at 37°C and then washed. BAM-conjugated anti-IFN-γ mAb (Mabtech) was added and plates were incubated for 2 hours at room temperature. After washing, anti-BAM 490 (Mabtech) was added and incubated for 1 hour at room temperature. The plates were dried and spot counting was performed using Iris fluorospot reader (Mabtech). The number of IFN-γ-producing cells in the control medium was subtracted from the number of IFN-γ producing cells in activated assays, and the results were expressed as spot number per 106 PBMCs.
7
Cytokine Profiling of GPR56+/- CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4 + T cells were divided into GPR56+ and GPR56-subsets, and these flow-sorted fractions (1-3 × 10 4 cells/well) and irradiated CD3-negative feeder cells (1:1) were cultured in precoated 96-well 3-color (IFN-γ, IL-10, IL-17A) FluoroSpot plates in the presence of heat denatured (70 °C, 1 hour) PR3-protein (5 μg/ml; Sigma Aldrich) or HCMV-glycoprotein-B (5 μg/ml; Advanced Targeting Systems Inc.) in RPMI1640 medium containing heat inactivated (56 °C, 30 minutes) 10% human serum (Sigma Aldrich) for 48 hours, protected from light and evaporation at 37 °C and 5% CO 2 . Next, cells were removed and plates were processed as per manufacturer instructions (Mabtech AB). Plates were read using the Mabtech Iris FluoroSpot reader. Readouts were assessed both as delta spot-forming units per million CD4+T cells and average relative spot volume (reflecting amount of cytokine secretion in a single spot). The mean + 2SD delta spot-forming unit in naïve GPR56-CD4+T cells was used as a threshold for a positive response in GPR56+CD4+T cells.
8
Quantifying SARS-CoV-2 T Cell Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify SARS-CoV-2 specific CD4 and CD8 T cells, peptide restimulation with Fluorospot for IFN and IL-2 was performed (Mabtech; Fluorospot Flex). In short, 2,5X10ˆ5 PBMC were resuspended in complete medium, plated in triplicate into 96 well plates with a PVDF membrane bottom layer, precoated overnight with capturing antibodies directed against IFN and IL-2. PBMC were stimulated with CD4 T cell (CD4-R and CD4-S MP) or CD8 T cell (CD8-A and CD8-B MP) specific peptide pools at a final concentration of 1 mg/mL, as described in Weiskopf et al.54 (link) After 23 hours of stimulation, plates were collected and spots were developed following manufacturer’s protocol (Mabtech, FSP-0102-10). Spots were revealed and quantified using Mabtech IRIS Fluorospot reader (Mabtech).
9
Quantifying Omicron-specific Immune Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IFN-γ ELISpot assays of mice were performed with a Mouse IFN-γ Precoated ELISpot Kit according to the manufacturer’s instructions (Dakewe). A total of 1 × 105 splenocytes per well were restimulated ex vivo with Omicron-BA.1 NTD–RBD peptide mix (2 μg/mL per peptide), negative control (DMSO), or positive control (50 ng/mL PMA + 1 μg/mL Innomycin), respectively.
IFN-γ ELISpot assays of 5 × 105 human PBMCs per well were performed with a Human IFN-γ Precoated ELISpot Kit purchased from Dakewe. Cells were restimulated ex vivo with Omicron-BA.1 NTD–RBD peptide mix (4 μg/mL per peptide), negative control (DMSO), or positive control (2.5 µg/mL PHA), respectively.
Twenty-four hours after restimulation, biotinylated antibody and streptavidin-HRP were added successively after cell lysis. Then, AEC peroxidase substrate was added, and spots were counted using an ELISpot plate reader (Mabtech IRIS™ Fluorospot Reader, Mabtech). Spot numbers were evaluated using Mabtech Apex™ software v.1.1.52.121.
IFN-γ ELISpot assays of 5 × 105 human PBMCs per well were performed with a Human IFN-γ Precoated ELISpot Kit purchased from Dakewe. Cells were restimulated ex vivo with Omicron-BA.1 NTD–RBD peptide mix (4 μg/mL per peptide), negative control (DMSO), or positive control (2.5 µg/mL PHA), respectively.
Twenty-four hours after restimulation, biotinylated antibody and streptavidin-HRP were added successively after cell lysis. Then, AEC peroxidase substrate was added, and spots were counted using an ELISpot plate reader (Mabtech IRIS™ Fluorospot Reader, Mabtech). Spot numbers were evaluated using Mabtech Apex™ software v.1.1.52.121.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!